Mechanism of Ivermectin Facilitation of Human P2X4 Receptor Channels

Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2X4 receptor channels (Khakh, B.S., W.R. Proctor, T.V. Dunwiddie, C. Labarca, and H.A. Lester. 1999. J. Neurosci. 19:7289–7299.). In the present study, the effects of IVM on the human P2X4 (hP2X4) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM (≤10 μM) applied from outside the cell (but not from inside) increased the maximum current activated by ATP, and slowed the rate of current deactivation. These two phenomena likely result from the binding of IVM to separate sites. A higher affinity site (EC50 0.25 μM) increased the maximal current activated by saturating concentrations of ATP without significantly changing the rate of current deactivation or the EC50 and Hill slope of the ATP concentration-response relationship. A lower affinity site (EC50 2 μM) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2X4 receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 μM) increased the number of openings per burst, without significantly changing the mean open or mean shut time within a burst. At higher concentrations (1.5 μM) of IVM, two additional open time components of long duration were observed that gave rise to long-lasting bursts of channel activity. Together, the results suggest that the binding of IVM to the higher affinity site increases current amplitude by reducing channel desensitization, whereas the binding of IVM to the lower affinity site slows the deactivation of the current predominantly by stabilizing the open conformation of the channel

Distinct Axonemal Processes Underlie Spontaneous and Stimulated Airway Ciliary Activity

Cilia are small organelles protruding from the cell surface that beat synchronously, producing biological transport. Despite intense research for over a century, the mechanisms underlying ciliary beating are still not well understood. Even the nature of the cytosolic molecules required for spontaneous and stimulated beating is debatable. In an effort to resolve fundamental questions related to cilia beating, we developed a method that integrates the whole-cell mode of the patch-clamp technique with ciliary beat frequency measurements on a single cell. This method enables to control the composition of the intracellular solution while the cilia remain intact, thus providing a unique tool to simultaneously investigate the biochemical and physiological mechanism of ciliary beating. Thus far, we investigated whether the spontaneous and stimulated states of cilia beating are controlled by the same intracellular molecular mechanisms. It was found that: (a) MgATP was sufficient to support spontaneous beating. (b) Ca2+ alone or Ca2+-calmodulin at concentrations as high as 1 μM could not alter ciliary beating. (c) In the absence of Ca2+, cyclic nucleotides produced a moderate rise in ciliary beating while in the presence of Ca2+ robust enhancement was observed. These results suggest that the axonemal machinery can function in at least two different modes

Controlling chaos in diluted networks with continuous neurons

Diluted neural networks with continuous neurons and nonmonotonic transfer function are studied, with both fixed and dynamic synapses. A noisy stimulus with periodic variance results in a mechanism for controlling chaos in neural systems with fixed synapses: a proper amount of external perturbation forces the system to behave periodically with the same period as the stimulus.Comment: 11 pages, 8 figure

Model of ionic currents through microtubule nanopores and the lumen

It has been suggested that microtubules and other cytoskeletal filaments may act as electrical transmission lines. An electrical circuit model of the microtubule is constructed incorporating features of its cylindrical structure with nanopores in its walls. This model is used to study how ionic conductance along the lumen is affected by flux through the nanopores when an external potential is applied across its two ends. Based on the results of Brownian dynamics simulations, the nanopores were found to have asymmetric inner and outer conductances, manifested as nonlinear IV curves. Our simulations indicate that a combination of this asymmetry and an internal voltage source arising from the motion of the C-terminal tails causes a net current to be pumped across the microtubule wall and propagate down the microtubule through the lumen. This effect is demonstrated to enhance and add directly to the longitudinal current through the lumen resulting from an external voltage source, and could be significant in amplifying low-intensity endogenous currents within the cellular environment or as a nano-bioelectronic device.Comment: 43 pages, 6 figures, revised versio

Correlations between hidden units in multilayer neural networks and replica symmetry breaking

We consider feed-forward neural networks with one hidden layer, tree architecture and a fixed hidden-to-output Boolean function. Focusing on the saturation limit of the storage problem the influence of replica symmetry breaking on the distribution of local fields at the hidden units is investigated. These field distributions determine the probability for finding a specific activation pattern of the hidden units as well as the corresponding correlation coefficients and therefore quantify the division of labor among the hidden units. We find that although modifying the storage capacity and the distribution of local fields markedly replica symmetry breaking has only a minor effect on the correlation coefficients. Detailed numerical results are provided for the PARITY, COMMITTEE and AND machines with K=3 hidden units and nonoverlapping receptive fields.Comment: 9 pages, 3 figures, RevTex, accepted for publication in Phys. Rev.

Intracellular Ca2+ Regulates the Phosphorylation and the Dephosphorylation of Ciliary Proteins Via the NO Pathway

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO syntase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating

Storage capacity of correlated perceptrons

We consider an ensemble of $K$ single-layer perceptrons exposed to random inputs and investigate the conditions under which the couplings of these perceptrons can be chosen such that prescribed correlations between the outputs occur. A general formalism is introduced using a multi-perceptron costfunction that allows to determine the maximal number of random inputs as a function of the desired values of the correlations. Replica-symmetric results for $K=2$ and $K=3$ are compared with properties of two-layer networks of tree-structure and fixed Boolean function between hidden units and output. The results show which correlations in the hidden layer of multi-layer neural networks are crucial for the value of the storage capacity.Comment: 16 pages, Latex2